An in vitro assay approach to investigate the potential impact of different doping agents on the steroid profile.

The steroid profile, that is, the urinary concentrations and concentration ratios of selected steroids, is used in sports drug testing to detect the misuse of endogenous steroids such as testosterone. Since several years, not only population-based thresholds are applied but also the steroid profile is monitored via the Athlete Biological Passport whereby the individual reference ranges derived from multiple test results of the same athlete are compared to population-based thresholds. In order to maintain a high probative force of the passport, samples collected or analyzed under suboptimal conditions should not be included in the longitudinal review. This applies to biologically affected or degraded samples and to samples excluded owing to the presence of other substances potentially (or evidently) altering the steroid profile. Nineteen different doping agents comprising anabolic steroids, selective androgen receptor modulators, selective estrogen receptor modulators, ibutamoren, and tibolone were investigated for their effect on the steroid profile using an androgen receptor activation test, an androgen receptor binding assay, an aromatase assay, and a steroidogenesis assay. The in vitro tests were coupled with well-established liquid chromatography/mass spectrometry-based analytical approaches and for a subset of steroidal analytes by gas chromatography/mass spectrometry. The variety of tests employed should produce a comprehensive data set to better understand how a compound under investigation may impact the steroid profile. Although our data set may allow an estimate of whether or not a substance will have an impact on the overall steroid metabolism, predicting which parameter in particular may be influenced remains difficult.

A novel co-culture assay to assess anti-tumor CD8+ T cell cytotoxicity via luminescence and multicolor flow cytometry.

T cell immunotherapies have shown great promise in patients with advanced cancer disease, revolutionizing treatment. T cell cytotoxicity is crucial in its efficacy, therefore developing ex vivo methods testing tumor and T cell interactions is pivotal. Increasing efforts have been made in developing co-culture assays with sophisticated materials and platforms aiming to mimic the tumor microenvironment (TME), but its complexity makes it difficult to develop the ideal model. In this study, we developed a simple co-culture assay, reproducible in any lab, but respecting the multicellular nature of the TME. Our goal is to combine in a single assay well-established techniques such as a luciferase assay for target cell viability analysis, a CD107a degranulation assay, and multicolor flow cytometry for the detection of cytokines and cytotoxicity markers. Cell suspensions of whole spleens and tumors containing splenic or tumor-infiltrating effector T cells of mice bearing Lewis lung carcinoma (LLC) or CT26 colon carcinoma tumors treated with radiation alone or in combination with immunotherapies were used for co-culture. LLC and CT26 cell lines transduced with the firefly luciferase gene were used as target cells. We demonstrated that splenocytes and tumor-infiltrating T cells derived from mice treated with combination therapy were able to kill approximately 50% of target cells after 48 h of co-culture. This effect was tumor cell-specific and dependent on CD8+ T cells evidenced by in vitro CD8+ T cell depletion. Flow cytometry demonstrated increased expression of CD107a and production of granzyme B, IFNγ, and TNFα by CD8+ T cells. Our co-culture assay is therefore suitable as proof of principle for in vivo therapeutic studies testing immunotherapies, and specifically to assess the involvement of cytotoxic CD8+ T cells in treatment response in LLC and CT26 tumor models. We also propose this assay as an ex vivo platform for high-throughput screening of immunomodulating agents to be tested in these two murine tumor models. This assay can be adapted to other tumor models after optimizations.

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis.

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable model for tracing tumor progression and angiogenesis simultaneously in a visual and sensitive manner. Bioluminescence imaging displays its unique superiority in living imaging due to its advantages of high sensitivity, strong specificity, and accurate measurement. Presented here is a protocol to establish a tumor-bearing mouse model by injecting a Renilla luciferase-labeled murine breast cancer cell line 4T1 into the transgenic mouse with angiogenesis-induced Firefly luciferase expression. This mouse model provides a valuable tool to simultaneously monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging in a single mouse. This model may be widely applied in anti-tumor drug screening and oncology research.

Biosynthetic bifunctional enzyme complex with high-efficiency luciferin-recycling to enhance the bioluminescence imaging.

Firefly luciferase is a prominent reporter on molecular imaging with the advantage of longer wavelength on light emission and the ATP linear correlation, which makes it useful in most of current bioluminescence imaging model. However, the utility of this biomaterial was limited by the signal intensity and stability which are respectively affected by enzyme activity and substrate consumption. This study demonstrated a series of novel synthetic bifunctional enzyme complex of Firefly luciferase (Fluc) and Luciferin-regenerating enzyme (LRE). A peptide linker library was constructed for the fusion strategy on biosynthesis. The findings of both experimental data and structural simulation demonstrated that the intervention of fused LRE remarkably improve the stability of in vitro bioluminescence signal through luciferin recycling; and revealed the competitive relationship of Fluc and LRE on luciferin binding: Fluc performed higher activity with one copy number of rigid linker (EAAAK) at the C terminal while LRE acted more efficiently with two copy numbers of flexible linker (GGGGS) at the N terminal. With the advantage of signal intensity and stability, this fused bifunctional enzyme complex may expand the application of firefly luciferase to in vitro bioluminescence imaging.

Editor's Highlight: Structure-Based Investigation on the Binding and Activation of Typical Pesticides With Thyroid Receptor.

A broad range of pesticides have been reported to interfere with the normal function of the thyroid endocrine system. However, the precise mechanism(s) of action has not yet been thoroughly elucidated. In this study, 21 pesticides were assessed for their binding interactions and the potential to disrupt thyroid homeostasis. In the GH3 luciferase reporter gene assays, 5 of the pesticides tested had agonistic effects in the order of procymidone > imidacloprid > mancozeb > fluroxypyr > atrazine. 11 pesticides inhibited luciferase activity of T3 to varying degrees, demonstrating their antagonistic activity. And there are 4 pesticides showed mixed effects when treated with different concentrations. Surface plasmon resonance (SPR) biosensor technique was used to directly measure the binding interactions of these pesticides to the human thyroid hormone receptor (hTR). 13 pesticides were observed to bind directly with TR, with a KD ranging from 4.80E-08 M to 9.44E-07 M. The association and disassociation of the hTR/pesticide complex revealed 2 distinctive binding modes between the agonists and antagonists. At the same time, a different binding mode was displayed by the pesticides showed mix agonist and antagonist activity. In addition, the molecular docking simulation analyses indicated that the interaction energy calculated by CDOCKER for the agonists and antagonists correlated well with the KD values measured by the surface plasmon resonance assay. These results help to explain the differences of the TR activities of these tested pesticides.

Development of a luciferase reporter Jurkat cell line under the control of endogenous interleukin-2 promoter.

During new drug development, it is critical to have a cell-based reporter bioassay to measure drug-mediated physiological changes. In a conventional reporter cell line, a reporter expression construct is randomly inserted into the host cell genome with the reporter gene under control of an engineered promoter. This design ensures high signal output but may not represent the true physiological cell signaling. Here we used the CRISPR/Cas9 technology to engineer a Jurkat cell line by replacing one interleukin 2 (IL2) allele with firefly luciferase gene while keeping the other IL2 allele intact. The expression of luciferase is thus under control of endogenous IL2 promoter. We demonstrated that, in this engineered cell line, the IL-2 secretion pathway remained intact and luciferase activity significantly increased upon stimulation with phorbol ester or CD3/CD28 antibodies. We next expressed glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) in this cell line and observed dose-dependent IL-2 and luciferase responses to GITR agonist antibody. Thus we have successfully constructed a reporter cell line by engineering a reporter gene under control of an endogenous target gene promoter. This novel strategy may provide a more physiologically relevant alternative to the traditional method of reporter cell line construction.

Circadian rhythms identified in Caenorhabditis elegans by in vivo long-term monitoring of a bioluminescent reporter.

Circadian rhythms are based on endogenous clocks that allow organisms to adjust their physiology and behavior by entrainment to the solar day and, in turn, to select the optimal times for most biological variables. Diverse model systems-including mice, flies, fungi, plants, and bacteria-have provided important insights into the mechanisms of circadian rhythmicity. However, the general principles that govern the circadian clock of Caenorhabditis elegans have remained largely elusive. Here we report robust molecular circadian rhythms in C elegans recorded with a bioluminescence assay in vivo and demonstrate the main features of the circadian system of the nematode. By constructing a luciferase-based reporter coupled to the promoter of the suppressor of activated let-60 Ras (sur-5) gene, we show in both population and single-nematode assays that C elegans expresses ∼24-h rhythms that can be entrained by light/dark and temperature cycles. We provide evidence that these rhythms are temperature-compensated and can be re-entrained after phase changes of the synchronizing agents. In addition, we demonstrate that light and temperature sensing requires the photoreceptors LITE and GUR-3, and the cyclic nucleotide-gated channel subunit TAX-2. Our results shed light on C elegans circadian biology and demonstrate evolutionarily conserved features in the circadian system of the nematode.

Nucleic acid clamp-mediated recognition and stabilization of the physiologically relevant MYC promoter G-quadruplex.

The MYC proto-oncogene is upregulated, often at the transcriptional level, in ∼80% of all cancers. MYC's promoter is governed by a higher order G-quadruplex (G4) structure in the NHE III1 region. Under a variety of conditions, multiple isoforms have been described to form from the first four continuous guanine runs (G41-4) predominating under the physiologically relevant supercoiled conditions. In the current study, short oligonucleotides complementing the 5'- and 3'-regions flanking the G4 have been connected by an abasic linker to form G4 clamps, varying both linker length and G4 isoform being targeted. Clamp A with an 18 Šlinker was found to have marked affinity for its target isomer (G41-4) over the other major structures (G42-5 and G41-5, recognized by clamps B and C, respectively), and to be able to shift equilibrating DNA to foster greater G4 formation. In addition, clamp A, but not B or C, is able to modulate MYC promoter activity with a significant and dose-dependent effect on transcription driven by the Del4 plasmid. This linked clamp-mediated approach to G4 recognition represents a novel therapeutic mechanism with specificity for an individual promoter structure, amenable to a large array of promoters.

The perplexities of the ZC3H12A self-mRNA regulation.

The mechanisms regulating transcript turnover are key processes in the regulation of gene expression. The list of proteins involved in mRNAs' degradation is still growing, however, the details of RNase-mRNAs interactions are not fully understood. ZC3H12A is a recently discovered inflammation-related RNase engaged in the control of proinflammatory cytokine transcript turnover. ZC3H12A also regulates its own transcript half-live. Here, we studied the details of this regulation. Our results confirm the importance of the 3'UTR in ZC3H12A-dependent ZC3H12A mRNA degradation. We compared the mouse and human stemloop structures present in this region and discovered that the human conserved stem-loop structure is not sufficient for ZC3H12A-dependent degradation. However, this structure is important for the ZC3H12A mRNA post-transcriptional regulation. Our studies emphasize the importance of the neighboring features of the identified stem-loop structure for its biological activity. Removal of this region together with the stem-loop structure greatly inhibits the ZC3H12A regulation of the investigated 3'-untranslated region (3'UTR).

Role of pulmonary macrophages in initiation of lung metastasis in anaplastic thyroid cancer.

Several clinical studies have demonstrated that increased macrophage infiltration into tumors confers metastatic potential and poor prognosis in cancer. Preclinical studies are needed to develop new strategies for countering metastasis. Our study was designed to investigate the impact of pulmonary macrophages on lung metastasis of anaplastic thyroid cancer (ATC). ATC (CAL-62) and macrophage (Raw264.7) were transfected with the effluc (CAL-62/effluc, Raw264.7/effluc). Coculture and migration assays were used to assess the effect of Raw264.7 or THP1 (human macrophage) (or conditioned medium) on the proliferation and/or migration of CAL-62/effluc cells in vitro. The effect of clodro-lipo or PBS-lipo on macrophage depletion was confirmed in vitro and in vivo. CAL-62/effluc cells (1 × 10(6) ) were intravenously injected into nude mice 24 h after clodro-lipo or PBS-lipo administration. Effect of clodro-lipo on the lung metastasis of CAL-62/effluc was assessed by bioluminescence imaging (BLI). Micro computed tomography (micro-CT) and histology. BLI signals of CAL-62/effluc and Raw264.7/effluc increased to cell number. Raw264.7 cells and THP1 cells promoted CAL-62/effluc proliferation, and conditioned medium of Raw264.7 cells promoted CAL-62/effluc migration. Clodro-lipo significantly depleted pulmonary macrophages in vitro and in vivo. Intensity of BLI signals in ATC lung metastasis was weaker in the clodro-lipo group than PBS-lipo control. Micro-CT imaging and hematoxylin/eosin staining revealed smaller tumor masses in the clodro-lipo group than PBS-lipo control. Our findings indicate that pulmonary macrophages have an important role in initiation of lung metastasis of ATC. New therapeutic strategies that preclude initiation of pulmonary metastasis could potentially be developed by targeting pulmonary macrophages.

An engineered tale-transcription factor rescues transcription of factor VII impaired by promoter mutations and enhances its endogenous expression in hepatocytes.

Tailored approaches to restore defective transcription responsible for severe diseases have been poorly explored. We tested transcription activator-like effectors fused to an activation domain (TALE-TFs) in a coagulation factor VII (FVII) deficiency model. In this model, the deficiency is caused by the -94C > G or -61T > G mutation, which abrogate the binding of Sp1 or HNF-4 transcription factors. Reporter assays in hepatoma HepG2 cells naturally expressing FVII identified a single TALE-TF (TF4) that, by targeting the region between mutations, specifically trans-activated both the variant (>100-fold) and wild-type (20-40-fold) F7 promoters. Importantly, in the genomic context of transfected HepG2 and transduced primary hepatocytes, TF4 increased F7 mRNA and protein levels (2- to 3-fold) without detectable off-target effects, even for the homologous F10 gene. The ectopic F7 expression in renal HEK293 cells was modestly affected by TF4 or by TALE-TF combinations. These results provide experimental evidence for TALE-TFs as gene-specific tools useful to counteract disease-causing promoter mutations.

Detection and Functional Analysis of Tumor-Derived LXR Ligands.

There is growing evidence highlighting the ability of nuclear receptors to control not only metabolism, but also inflammation and cancer progression. In particular liver X receptors (LXRs), the nuclear receptors physiologically involved in cholesterol homeostasis, have been shown to regulate innate and adaptive immune responses in many pathological conditions, including cancer.We have recently demonstrated that LXR ligands (oxysterols) released by tumor cells may have an immunomodulatory role, affecting the immune cells involved in the antitumor immune response. Indeed, oxysterols inhibit the expression of the chemokine receptor CCR7 on dendritic cells (DC) in an LXR-dependent manner, thus impairing DC migration to secondary lymphoid organs, and therefore dampening the induction of successful antitumor responses.We have resorted to direct (i.e., luciferase-based LXR activation assay) and indirect (i.e., activation of LXR target genes in dendritic cells) methods in order to assess the presence of LXR ligands (oxysterols) in tumor-conditioned media.These two methods are also suitable to study strategies to block oxysterol release by tumor cells.

Inter-polysomal coupling of termination and initiation during translation in eukaryotic cell-free system.

The recording of the luciferase-generated luminescence in the eukaryotic cell-free translation system programmed with mRNA encoding firefly luciferase (Luc-mRNA) showed that the addition of free exogenous mRNAs into the translation reactor induces the immediate release of the functionally active protein from the polyribosomes of the translation system. The phenomenon did not depend on the coding specificity of the added free mRNA. At the same time it depended on the "initiation potential" of the added mRNA (including the features that ensure the successful initiation of translation, such as the presence of the cap structure and the sufficient concentration of the added mRNA in the translation mixture). The phenomenon also strictly depended on the presence of the stop codon in the translated mRNA. As the above-mentioned features of the added mRNA imply its activity in initiation of a new translation, the experimental data are found in agreement with the scenario where the molecules of the added mRNA interact by their 5'-ends with terminating and recycling ribosomes, stimulating the release of the complete polypeptides and providing for the initiation of a new translation.

Rational design of novel N-alkyl-N capped biostable RNA nanostructures for efficient long-term inhibition of gene expression.

Computational techniques have been used to design a novel class of RNA architecture with expected improved resistance to nuclease degradation, while showing interference RNA activity. The in silico designed structure consists of a 24-29 bp duplex RNA region linked on both ends by N-alkyl-N dimeric nucleotides (BCn dimers; n = number of carbon atoms of the alkyl chain). A series of N-alkyl-N capped dumbbell-shaped structures were efficiently synthesized by double ligation of BCn-loop hairpins. The resulting BCn-loop dumbbells displayed experimentally higher biostability than their 3'-N-alkyl-N linear version, and were active against a range of mRNA targets. We studied first the effect of the alkyl chain and stem lengths on RNAi activity in a screen involving two series of dumbbell analogues targeting Renilla and Firefly luciferase genes. The best dumbbell design (containing BC6 loops and 29 bp) was successfully used to silence GRB7 expression in HER2+ breast cancer cells for longer periods of time than natural siRNAs and known biostable dumbbells. This BC6-loop dumbbell-shaped structure displayed greater anti-proliferative activity than natural siRNAs.

Selective Estrogen Receptor Modulators and the Tissue-Selective Estrogen Complex: Analysis of Cell Type-Specific Effects Using In Vivo Imaging of a Reporter Mouse Model.

Selective estrogen receptor modulators (SERMs) are a class of compounds that act differentially on the estrogen receptor (ER) in various tissues with a mixed agonist/antagonistic activity (agonistic in some tissues while antagonist in others). This peculiarity represents a challenge for developing new hormone replacement therapies (HRTs) and highlights the need of new tools to evaluate the specific effects of a given SERM in different organs/tissues of an entire organism and with time. Reporter mice represent invaluable tools in pharmacology to analyze specific signaling in physiological conditions and monitor the effects of drugs acting on these signals in a spatio-temporal dimension. Here, we describe an in vivo protocol to examine the effects of different SERMs on estrogen receptor activity by using the ERE-Luc reporter model, a mouse that reports ER transcriptional activity.

Different Types of Luciferase Reporters Show Distinct Susceptibility to T3-Evoked Downregulation.

BACKGROUND: The firefly luciferase reporter protein is a crucial tool for studies targeting a broad range of biological questions. Importantly, luciferase assays are also widely used to explore mechanisms underlying thyroid hormone dependent regulation of gene expression. However, it was demonstrated that the firefly luciferase reporter is subject to triiodothyronine (T3)-evoked, promoter independent downregulation that is mediated by the thyroid hormone receptor. Since this effect can interfere with readout accuracy, the study aimed to find luciferase reporters that are not susceptible to this phenomenon. METHODS: Luciferase reporter constructs were generated under the control of a minimal thymidine kinase (TK) promoter and transiently transfected into JEG-3 cells to test their activity upon T3 treatment. RESULTS: Activity of the TK-(dCpG)Luc encoding a synthetic (dCpG)Luciferase and TK-NanoLuc expressing the NanoLuc reporter was not significantly changed by T3 treatment while the firefly luciferase control was suppressed by ∼2.6-fold. T3 also downregulated the activity of Renilla luciferase by ∼30%. CONCLUSIONS: Novel types of luciferase reporters, especially the synthetic (dCpG)Luciferase, can be more accurate to study T3-regulated gene expression than the classical firefly luciferase reporter. Renilla luciferase, a popular transfection control of dual luciferase assays, should be used with caution in conditions with T3 treatment.

Photochemical Regulation of Gene Expression Using Caged siRNAs with Single Terminal Vitamin†E Modification.

Caged siRNAs with a single photolabile linker and/or vitamin E (vitE) modification at the 5' terminal were rationally designed and synthesized. These virtually inactive caged siRNAs were successfully used to photoregulate both firefly luciferase and GFP gene expression in cells with up to an 18.6-fold enhancement of gene silencing activity, which represents one of the best reported photomodulation of gene silencing efficiencies to date. siRNA tracking and vitE competition experiments indicated that the inactivity of vitE-modified siRNAs was not due to the bulky moiety of vitE; rather, the involvement of vitE-binding proteins has a large contribution to caged siRNA inactivation by preventing the dissociation of siRNA/lipo complexes and/or siRNA release. Further patterning experiments revealed the ability to spatially regulate gene expression through simple light irradiation.

Enhancement of adoptive T cell transfer with single low dose pretreatment of doxorubicin or paclitaxel in mice.

Ex vivo expansion of CD8+ T-cells has been a hindrance for the success of adoptive T cell transfer in clinic. Currently, preconditioning with chemotherapy is used to modulate the patient immunity before ACT, however, the tumor microenvironment beneficial for transferring T cells may also be damaged. Here preconditioning with single low dose of doxorubicin or paclitaxel combined with fewer CD8+ T-cells was investigated to verify whether the same therapeutic efficacy of ACT could be achieved. An E.G7/OT1 animal model that involved adoptive transfer of OVA-specific CD8+ T-cells transduced with a granzyme B promoter-driven firefly luciferase and tomato fluorescent fusion reporter gene was used to evaluate this strategy. The result showed that CD8+ T-cells were activated and sustained longer in mice pretreated with one low-dose Dox or Tax. Enhanced therapeutic efficacy was found in Dox or Tax combined with 2x106 CD8+ T-cells and achieved the same level of tumor growth inhibition as that of 5x106 CD8+ T-cells group. Notably, reduced numbers of Tregs and myeloid derived suppressor cells were shown in combination groups. By contrast, the number of tumor-infiltrating cytotoxic T lymphocytes and IL-12 were increased. The NF-κB activity and immunosuppressive factors such as TGF-ß, IDO, CCL2, VEGF, CCL22, COX-2 and IL-10 were suppressed. This study demonstrates that preconditioning with single low dose Dox or Tax and combined with two fifth of the original CD8+ T-cells could improve the tumor microenvironment via suppression of NF-κB and its related immunosuppressors, and activate more CD8+ T-cells which also stay longer.

Translational misreading in Mycobacterium smegmatis increases in stationary phase.

The study of errors in gene translation has largely been confined to a small number of model organisms. We have examined all possible misreading errors at a defined codon in Mycobacterium smegmatis. Using a dual-luciferase gain of function reporter system that employs a mutated essential lysine in firefly luciferase, we accurately quantified mistranslation errors. Overall, accuracy of gene translation was comparable with Escherichia coli at <1/2000 errors/codon during exponential growth. Stationary phase was associated with a dramatic increase in misincorporation errors by Lys-tRNACUU(Lys) at a subset of three codons, each with a single base changed from the AAG lysine codon. The maximum error rate detected was 0.2% with codon AUG. Treatment with streptomycin increased misreading errors at several codons associated in particular with U·U, G·U and C·U codon·anti-codon mismatches, but oxidative stress did not change translational fidelity. Our study is the first comprehensive examination of misreading errors for a defined codon in mycobacteria.

VEGF therapeutic gene delivery using dendrimer type bio-reducible polymer into human mesenchymal stem cells (hMSCs).

The therapeutic potential of mesenchymal stem cells (MSCs) has garnered great attention in the expansive diversity of biomedical research. Despite this broad interest in stem cells, limited incorporation and poor viability are major disadvantages for accomplishing therapeutic success in the field of hMSC-based cell therapy, and an optimal approach for hMSC-based cell therapy using non-viral vectors has not been established. Hence, we examined the possibility of performing gene therapy using the biodegradable polymeric non-viral vector Arginine-grafted poly (cystaminebisacrylamide-diaminohexane) (ABP)-conjugated poly (amidoamine) (PAMAM) dendrimer (PAM-ABP) in hMSCs. PAM-ABP formed compact nanosized polyplexes and showed low cytotoxicity compared to bPEI 25k and Lipofectamine® 2000 in hMSCs. Although the cellular uptake was similar, the transfection efficiency and VEGF expression of PAM-ABP using gWiz-Luc and pß-VEGF were higher than those of the control groups. Although hMSCs were transfected, their stem cell characteristics were retained. Our results suggest that PAM-ABP has the ability to deliver a therapeutic gene in hMSCs.